Allografts combined with tissue derived stem cells for bone healing

ABSTRACT

There is disclosed a method of combining mesenchymal stem cells (MSCs) with a bone substrate. In an embodiment, the method includes obtaining tissue having MSCs together with unwanted cells. The tissue is digested to form a cell suspension having MSCs and unwanted cells. The cell suspension is added to the substrate. The substrate is cultured to allow the MSCs to adhere. The substrate is rinsed to remove unwanted cells. In various embodiments, the tissue is adipose issue, muscle tissue, or bone marrow tissue. In an embodiment, there is disclosed an allograft product including a combination of MSCs with a bone substrate in which the combination is manufactured by culturing MSCs disposed on the substrate for a period of time to allow the MSCs to adhere to the substrate, and then rinsing the substrate to remove unwanted cells from the substrate. Other embodiments are also disclosed.

REFERENCE TO PENDING PRIOR PATENT APPLICATION

This application claims the benefit under 35 U.S.C. 119 (e) of U.S.Provisional Patent Application No. 61/116,484, filed Nov. 20, 2008 byYaling Shi for “ALLOGRAFTS COMBINED WITH TISSUE DERIVED STEM CELLS FORBONE HEALING,” which patent application is hereby incorporated herein byreference.

BACKGROUND

Regenerative medicine requires an abundant source of human adult stemcells that can be readily available at the point of care.

Adipose-derived stem cells (ASCs), which can be obtained in largequantities, have been utilized as cellular therapy for the induction ofbone formation in tissue engineering strategies.

Allografts may be combined with stem cells. This requires a significantamount of tissue processing and cellular processing prior to seeding theallograft substrate.

Allografts seeded with living cells generally provide better surgicalresults.

SUMMARY OF THE INVENTION

In an embodiment, there is provided a method of combining mesenchymalstem cells with a bone substrate, the method comprising obtainingadipose tissue having the mesenchymal stem cells together with unwantedcells; digesting the adipose tissue to form a cell suspension having themesenchymal stem cells and the unwanted cells; adding the cellsuspension with the mesenchymal stem cells to seed the bone substrate soas to form a seeded bone substrate; culturing the mesenchymal stem cellson the seeded bone substrate for a period of time to allow themesenchymal stem cells to adhere to the bone substrate; and rinsing thebone substrate to remove the unwanted cells from the bone substrate.

In another embodiment, there is provided an allograft product includinga combination of mesenchymal stem cells with a bone substrate, and thecombination manufactured by obtaining adipose tissue having themesenchymal stem cells together with unwanted cells; digesting theadipose tissue to form a cell suspension having the mesenchymal stemcells and the unwanted cells; adding the cell suspension with themesenchymal stem cells to seed the bone substrate so as to form a seededbone substrate; culturing the mesenchymal stem cells on the seeded bonesubstrate for a period of time to allow the mesenchymal stem cells toadhere to the bone substrate; and rinsing the bone substrate to removethe unwanted cells from the bone substrate.

In still another embodiment, there is provided a method of combiningmesenchymal stem cells with a bone substrate, the method comprisingobtaining adipose tissue having the mesenchymal stem cells together withunwanted cells; digesting the adipose tissue to form a cell suspensionhaving the mesenchymal stem cells and the unwanted cells to acquire astromal vascular fraction, and the digesting includes making acollagenase I solution, and filtering the solution through a 0.2 μmfilter unit, mixing the adipose solution with the collagenase Isolution, and adding the adipose solution mixed with the collagenase Isolution to a shaker flask; placing the shaker with continuous agitationat about 75 RPM for about 45 to 60 minutes so as to provide the adiposetissue with a visually smooth appearance; aspirating a supernatantcontaining mature adipocytes so as to provide a pellet; adding the cellsuspension with the mesenchymal stem cells to seed the bone substrate soas to form a seeded bone substrate; culturing the mesenchymal stem cellson the seeded bone substrate for a period of time to allow themesenchymal stem cells to adhere to the bone substrate; and rinsing thebone substrate to remove the unwanted cells from the bone substrate.

In yet another embodiment, there is provided an allograft productincluding a combination of mesenchymal stem cells with a bone substrate,and the combination manufactured by obtaining adipose tissue having themesenchymal stem cells together with unwanted cells; digesting theadipose tissue to form a cell suspension having the mesenchymal stemcells and the unwanted cells to acquire a stromal vascular fraction, andthe digesting includes making a collagenase I solution, and filteringthe solution through a 0.2 μm filter unit, mixing the adipose solutionwith the collagenase I solution, and adding the adipose solution mixedwith the collagenase I solution to a shaker flask; placing the shakerwith continuous agitation at about 75 RPM for about 45 to 60 minutes soas to provide the adipose tissue with a visually smooth appearance;aspirating a supernatant containing mature adipocytes so as to provide apellet; adding the cell suspension with the mesenchymal stem cells toseed the bone substrate by adding the pellet onto the bone substrate soas to form a seeded bone substrate; culturing the mesenchymal stem cellson the seeded bone substrate for a period of time to allow themesenchymal stem cells to adhere to the bone substrate; and rinsing thebone substrate to remove the unwanted cells from the bone substrate.

In an embodiment, there is provided a method of combining mesenchymalstem cells with a bone substrate, the method comprising obtaining tissuehaving the mesenchymal stem cells together with unwanted cells;digesting the tissue to form a cell suspension having the mesenchymalstem cells and the unwanted cells; adding the cell suspension with themesenchymal stem cells to seed the bone substrate so as to form a seededbone substrate; culturing the mesenchymal stem cells on the seeded bonesubstrate for a period of time to allow the mesenchymal stem cells toadhere to the bone substrate; and rinsing the bone substrate to removethe unwanted cells from the bone substrate.

In another embodiment, there is provided an allograft product includinga combination of mesenchymal stem cells with a bone substrate, and thecombination manufactured by obtaining tissue having the mesenchymal stemcells together with unwanted cells; digesting the tissue to form a cellsuspension having the mesenchymal stem cells and the unwanted cells;adding the cell suspension with the mesenchymal stem cells to seed thebone substrate so as to form a seeded bone substrate; culturing themesenchymal stem cells on the seeded bone substrate for a period of timeto allow the mesenchymal stem cells to adhere to the bone substrate; andrinsing the bone substrate to remove the unwanted cells from the bonesubstrate.

In still another embodiment, there is provided a method of combiningmesenchymal stem cells with a bone substrate, the method comprisingobtaining bone marrow tissue having the mesenchymal stem cells togetherwith unwanted cells; digesting the bone marrow tissue to form a cellsuspension having the mesenchymal stem cells and the unwanted cells;adding the cell suspension with the mesenchymal stem cells to seed thebone substrate so as to form a seeded bone substrate; culturing themesenchymal stem cells on the seeded bone substrate for a period of timeto allow the mesenchymal stem cells to adhere to the bone substrate; andrinsing the bone substrate to remove the unwanted cells from the bonesubstrate.

In yet another embodiment, there is provided an allograft productincluding a combination of mesenchymal stem cells with a bone substrate,and the combination manufactured by obtaining bone marrow tissue havingthe mesenchymal stem cells together with unwanted cells; digesting thebone marrow tissue to form a cell suspension having the mesenchymal stemcells and the unwanted cells; adding the cell suspension with themesenchymal stem cells to seed the bone substrate so as to form a seededbone substrate; culturing the mesenchymal stem cells and the bonesubstrate for a period of time to allow the mesenchymal stem cells toadhere to the bone substrate; and rinsing the bone substrate to removethe unwanted cells from the bone substrate.

In an embodiment, there is provided a method of combining mesenchymalstem cells with a bone substrate, the method comprising obtaining muscletissue having the mesenchymal stem cells together with unwanted cells;digesting the muscle tissue to form a cell suspension having themesenchymal stem cells the unwanted cells; adding the cell suspensionwith the mesenchymal stem cells to seed the bone substrate so as to forma seeded bone substrate; culturing the mesenchymal stem cells on theseeded bone substrate for a period of time to allow the mesenchymal stemcells to adhere to the bone substrate; and rinsing the bone substrate toremove the unwanted cells from the bone substrate.

In another embodiment, there is provided an allograft product includinga combination of mesenchymal stem cells with a bone substrate, and thecombination manufactured by obtaining muscle tissue having themesenchymal stem cells together with unwanted cells; digesting themuscle tissue to form a cell suspension having the mesenchymal stemcells and the unwanted cells; adding the cell suspension with themesenchymal stem cells to seed the bone substrate so as to form a seededbone substrate; culturing the mesenchymal stem cells on the seeded bonesubstrate for a period of time to allow the mesenchymal stem cells toadhere to the bone substrate; and rinsing the bone substrate to removethe unwanted cells from the bone substrate.

Other embodiments are also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

Illustrative embodiments of the invention are illustrated in thedrawings, in which:

FIG. 1 illustrates a flow chart of the combination of mesenchymal stemcells with a bone substrate;

FIG. 2 illustrates a prior art example of a pellet of a stromal vascularfraction containing the desired stem cells and unwanted cells;

FIG. 3 illustrates various examples of strips (FIGS. 3A and 3B) anddowels (FIGS. 3C and 3D) which have a 3-D cancellous matrix structureand mesenchymal stem cells (MSCs) may adhere to;

FIG. 4 illustrates a standard curve of total live ASCs using the CCK-8assay;

FIG. 5 illustrates mineral deposition by ASCs cultured in osteogenicmedium; and

FIG. 6 illustrates H&E staining showed that cells adhered to the bonesurface.

DETAILED DESCRIPTION

Unless otherwise described, human adult stem cells are generallyreferred to as mesenchymal stem cells or MSCs. MSCs are pluripotentcells that have the capacity to differentiate in accordance with atleast two discrete development pathways. Adipose-derived stem cells orASCs are stem cells that are derived from adipose tissue. StromalVascular Fraction or SVF generally refers to the centrifuged cell pelletobtained after digestion of tissue containing MSCs. In one embodiment,the pellet may include multiple types of stem cells. These stem cellsmay include, for example, one or more of hematopoietic stem cells,epithelial progenitor cells, and mesenchymal stem cells. In anembodiment, mesenchymal stem cells are filtered from other stem cells bytheir adherence to a bone substrate, while the other stem cells (i.e.,unwanted cells) do not adhere to the bone substrate. Other cells that donot adhere to the bone substrate may also be included in these unwantedcells.

Adipose derived stem cells may be isolated from cadavers andcharacterized using flow cytometry and tri-lineage differentiation(osteogenesis, chondrogenesis and adipogenesis) may be performed invitro. The final product may be characterized using histology formicrostructure and biochemical assays for cell count. This consistentcell-based product may be useful for bone regeneration.

Tissue engineering and regenerative medicine approaches offer greatpromise to regenerate bodily tissues. The most widely studied tissueengineering approaches, which are based on seeding and in vitroculturing of cells within the scaffold before implantation, is the cellsource and the ability to control cell proliferation anddifferentiation. Many researchers have demonstrated that adiposetissue-derived stem cells (ASCs) possess multiple differentiationcapacities. See, for example, the following, which are incorporated byreference:

-   Rada, T., R. L. Reis, and M. E. Gomes, Adipose Tissue-Derived Stem    Cells and Their Application in Bone and Cartilage Tissue    Engineering. Tissue Eng Part B Rev, 2009.-   Ahn, N. H., et al., In vivo osteogenic differentiation of human    adipose-derived stem cells in an injectable in situ-forming gel    scaffold. Tissue Eng Part A, 2009. 15(7): p. 1821-32.-   Anghileri, E., et al., Neuronal differentiation potential of human    adipose-derived mesenchymal stem cells. Stem Cells Dev, 2008.    17(5): p. 909-16.-   Arnalich-Montiel, F., et al., Adipose-derived stem cells are a    source for cell therapy of the corneal stroma. Stem Cells, 2008.    26(2): p. 570-9.-   Bunnell, B. A., et al., Adipose-derived stem cells: isolation,    expansion and differentiation. Methods, 2008. 45(2): p. 115-20.-   Chen, R. B., et al., [Differentiation of rat adipose-derived stem    cells into smooth-muscle-like cells in vitro]. Zhonghua Nan Ke    Xue, 2009. 15(5): p. 425-30.-   Cheng, N. C., et al., Chondrogenic differentiation of    adipose-derived adult stem cells by a porous scaffold derived from    native articular cartilage extracellular matrix. Tissue Eng Part    A, 2009. 15(2): p. 231-41.-   Cui, L., et al., Repair of cranial bone defects with adipose derived    stem cells and coral scaffold in a canine model. Biomaterials, 2007.    28(36): p. 5477-86.-   de Girolamo, L., et al., Osteogenic differentiation of human    adipose-derived stem cells: comparison of two different inductive    media. J Tissue Eng Regen Med, 2007. 1(2): p. 154-7.-   Elabd, C., et al., Human adipose tissue-derived multipotent stem    cells differentiate in vitro and in vivo into osteocyte-like cells.    Biochem Biophys Res Commun, 2007. 361(2): p. 342-8.-   Flynn, L., et al., Adipose tissue engineering with naturally derived    scaffolds and adipose-derived stem cells. Biomaterials, 2007.    28(26): p. 3834-42.-   Flynn, L. E., et al., Proliferation and differentiation of    adipose-derived stem cells on naturally derived scaffolds.    Biomaterials, 2008. 29(12): p. 1862-71.-   Fraser, J. K., et al., Adipose-derived stem cells. Methods Mol Biol,    2008.449: p. 59-67.-   Gimble, J. and F. Guilak, Adipose-derived adult stem cells:    isolation, characterization, and differentiation potential.    Cytotherapy, 2003. 5(5): p. 362-9.-   Gimble, J. M. and F. Guilak, Differentiation potential of adipose    derived adult stem (ADAS) cells. Curr Top Dev Biol, 2003. 58: p.    137-60.-   Jin, X. B., et al., Tissue engineered cartilage from hTGF beta2    transduced human adipose derived stem cells seeded in PLGA/alginate    compound in vitro and in vivo. J Biomed Mater Res A, 2008. 86(4): p.    1077-87.-   Kakudo, N., et al., Bone tissue engineering using human    adipose-derived stem cells and honeycomb collagen scaffold. J Biomed    Mater Res A, 2008. 84(1): p. 191-7.-   Kim, H. J. and G. I. Im, Chondrogenic differentiation of adipose    tissue-derived mesenchymal stem cells: greater doses of growth    factor are necessary. J Orthop Res, 2009. 27(5): p. 612-9.-   Kingham, P. J., et al., Adipose-derived stem cells differentiate    into a Schwann cell phenotype and promote neurite outgrowth in    vitro. Exp Neurol, 2007. 207(2): p. 267-74.-   Mehlhorn, A. T., et al., Chondrogenesis of adipose-derived adult    stem cells in a poly-lactide-co-glycolide scaffold. Tissue Eng Part    A, 2009. 15(5): p. 1159-67.-   Merceron, C., et al., Adipose-derived mesenchymal stem cells and    biomaterials for cartilage tissue engineering. Joint Bone    Spine, 2008. 75(6): p. 672-4.-   Mischen, B. T., et al., Metabolic and functional characterization of    human adipose-derived stem cells in tissue engineering. Plast    Reconstr Surg, 2008. 122(3): p. 725-38.-   Mizuno, H., Adipose-derived stem cells for tissue repair and    regeneration: ten years of research and a literature review. J    Nippon Med Sch, 2009. 76(2): p. 56-66.-   Tapp, H., et al., Adipose-Derived Stem Cells: Characterization and    Current Application in Orthopaedic Tissue Repair. Exp Biol Med    (Maywood), 2008.-   Tapp, H., et al., Adipose-derived stem cells: characterization and    current application in orthopaedic tissue repair. Exp Biol Med    (Maywood), 2009. 234(1): p. 1-9.-   van Dijk, A., et al., Differentiation of human adipose-derived stem    cells towards cardiomyocytes is facilitated by laminin. Cell Tissue    Res, 2008. 334(3): p. 457-67.-   Wei, Y., et al., A novel injectable scaffold for cartilage tissue    engineering using adipose-derived adult stem cells. J Orthop    Res, 2008. 26(1): p. 27-33.-   Wei, Y., et al., Adipose-derived stem cells and chondrogenesis.    Cytotherapy, 2007. 9(8): p. 712-6.-   Zhang, Y. S., et al., [Adipose tissue engineering with human    adipose-derived stem cells and fibrin glue injectable scaffold].    Zhonghua Yi Xue Za Zhi, 2008. 88(38): p. 2705-9.

Additionally, adipose tissue is probably the most abundant andaccessible source of adult stem cells. Adipose tissue derived stem cellshave great potential for tissue regeneration. Nevertheless, ASCs andbone marrow-derived stem cells (BMSCs) are remarkably similar withrespect to growth and morphology, displaying fibroblasticcharacteristics, with abundant endoplasmic reticulum and large nucleusrelative to the cytoplasmic volume. See, for example, the following,which are incorporated by reference:

-   Gimble, J. and F. Guilak, Adipose-derived adult stem cells:    isolation, characterization, and differentiation potential.    Cytotherapy, 2003. 5(5): p. 362-9.-   Gimble, J. M. and F. Guilak, Differentiation potential of adipose    derived adult stem (ADAS) cells. Curr Top Dev Biol, 2003. 58: p.    137-60.-   Strem, B. M., et al., Multipotential differentiation of adipose    tissue-derived stem cells. Keio J Med, 2005. 54(3): p. 132-41.-   De Ugarte, D. A., et al., Comparison of multi-lineage cells from    human adipose tissue and bone marrow. Cells Tissues Organs, 2003.    174(3): p. 101-9.-   Hayashi, O., et al., Comparison of osteogenic ability of rat    mesenchymal stem cells from bone marrow, periosteum, and adipose    tissue. Calcif Tissue Int, 2008. 82(3): p. 238-47.-   Kim, Y., et al., Direct comparison of human mesenchymal stem cells    derived from adipose tissues and bone marrow in mediating    neovascularization in response to vascular ischemia. Cell Physiol    Biochem, 2007. 20(6): p. 867-76.-   Lin, L., et al., Comparison of osteogenic potentials of BMP4    transduced stem cells from autologous bone marrow and fat tissue in    a rabbit model of calvarial defects. Calcif Tissue Int, 2009.    85(1): p. 55-65.-   Niemeyer, P., et al., Comparison of immunological properties of bone    marrow stromal cells and adipose tissue-derived stem cells before    and after osteogenic differentiation in vitro. Tissue Eng, 2007.    13(1): p. 111-21.-   Noel, D., et al., Cell specific differences between human    adipose-derived and mesenchymal-stromal cells despite similar    differentiation potentials. Exp Cell Res, 2008. 314(7): p. 1575-84.-   Yoo, K. H., et al., Comparison of immunomodulatory properties of    mesenchymal stem cells derived from adult human tissues. Cell    Immunol, 2009.-   Yoshimura, H., et al., Comparison of rat mesenchymal stem cells    derived from bone marrow, synovium, periosteum, adipose tissue, and    muscle. Cell Tissue Res, 2007. 327(3): p. 449-62.

Other common characteristics of ASCs and BMSCs can be found in thetranscriptional and cell surface profile. Several studies have alreadybeen done in the field of bone tissue engineering using ASCs. See, forexample, the following, which are incorporated by reference:

-   Rada, T., R. L. Reis, and M. E. Gomes, Adipose Tissue-Derived Stem    Cells and Their Application in Bone and Cartilage Tissue    Engineering. Tissue Eng Part B Rev, 2009.-   Tapp, H., et al., Adipose-Derived Stem Cells: Characterization and    Current Application in Orthopaedic Tissue Repair. Exp Biol Med    (Maywood), 2008.-   Tapp, H., et al., Adipose-derived stem cells: characterization and    current application in orthopaedic tissue repair. Exp Biol Med    (Maywood), 2009. 234(1): p. 1-9.-   De Girolamo, L., et al., Human adipose-derived stem cells as future    tools in tissue regeneration: osteogenic differentiation and    cell-scaffold interaction. Int J Artif Organs, 2008. 31(6): p.    467-79.-   Di Bella, C., P. Farlie, and A. J. Penington, Bone regeneration in a    rabbit critical-sized skull defect using autologous adipose-derived    cells. Tissue Eng Part A, 2008. 14(4): p. 483-90.-   Grewal, N. S., et al., BMP-2 does not influence the osteogenic fate    of human adipose-derived stem cells. Plast Reconstr Surg, 2009.    123(2 Suppl): p. 158S-65S.-   Li, H., et al., Bone regeneration by implantation of adipose-derived    stromal cells expressing BMP-2. Biochem Biophys Res Commun, 2007.    356(4): p. 836-42.-   Yoon, E., et al., In vivo osteogenic potential of human    adipose-derived stem cells/poly lactide-co-glycolic acid constructs    for bone regeneration in a rat critical-sized calvarial defect    model. Tissue Eng, 2007. 13(3): p. 619-27.

These studies have demonstrated that stem cells obtained from theadipose tissue exhibit good attachment properties to most of thematerial surfaces and the capacity to differentiate intoosteoblastic-like cells in vitro and in vivo. Recently it has been shownthat ASCs may stimulate the vascularization process. See, for example,the following, which are incorporated by reference:

-   Butt, O. I., et al., Stimulation of peri-implant vascularization    with bone marrow-derived progenitor cells: monitoring by in vivo EPR    oximetry. Tissue Eng, 2007. 13(8): p. 2053-61.-   Rigotti, G., et al., Clinical treatment of radiotherapy tissue    damage by lipoaspirate transplant: a healing process mediated by    adipose-derived adult stem cells. Plast Reconstr Surg, 2007.    119(5): p. 1409-22; discussion 1423-4.    Demineralized bone substrate, as an allogeneic material, is a    promising bone tissue-engineering scaffold due to its close relation    to autologous bone in terms of structure and function. Combined with    MSCs, these scaffolds have been demonstrated to accelerate and    enhance bone formation within osseous defects when compared with the    matrix alone. See, for example, the following, which are    incorporated by reference:-   Chen, L. Q., et al., [Study of MSCs in vitro cultured on    demineralized bone matrix of mongrel]. Shanghai Kou Qiang Yi    Xue, 2007. 16(3): p. 255-8.-   Gamradt, S. C. and J. R. Lieberman, Bone graft for revision hip    arthroplasty: biology and future applications. Clin Orthop Relat    Res, 2003(417): p. 183-94.-   Honsawek, S., D. Dhitiseith, and V. Phupong, Effects of    demineralized bone matrix on proliferation and osteogenic    differentiation of mesenchymal stem cells from human umbilical cord.    J Med Assoc That, 2006. 89 Suppl 3: p. S189-95.-   Kasten, P., et al., [Induction of bone tissue on different matrices:    an in vitro and a in vivo pilot study in the SCID mouse]. Z Orthop    Ihre Grenzgeb, 2004. 142(4): p. 467-75.-   Kasten, P., et al., Ectopic bone formation associated with    mesenchymal stem cells in a resorbable calcium deficient    hydroxyapatite carrier. Biomaterials, 2005. 26(29): p. 5879-89.-   Qian, Y., Z. Shen, and Z. Zhang, [Reconstruction of bone using    tissue engineering and nanoscale technology]. Zhongguo Xiu Fu Chong    Jian Wai Ke Za Zhi, 2006. 20(5): p. 560-4.-   Reddi, A. H., Role of morphogenetic proteins in skeletal tissue    engineering and regeneration. Nat Biotechnol, 1998. 16(3): p.    247-52.-   Reddi, A. H., Morphogenesis and tissue engineering of bone and    cartilage: inductive signals, stem cells, and biomimetic    biomaterials. Tissue Eng, 2000. 6(4): p. 351-9.-   Tsiridis, E., et al., In vitro and in vivo optimization of impaction    allografting by demineralization and addition of rh-OP-1. J Orthop    Res, 2007. 25(11): p. 1425-37.-   Xie, H., et al., The performance of a bone-derived scaffold material    in the repair of critical bone defects in a rhesus monkey model.    Biomaterials, 2007. 28(22): p. 3314-24.-   Liu, G., et al., Tissue-engineered bone formation with cryopreserved    human bone marrow mesenchymal stem cells. Cryobiology, 2008.    56(3): p. 209-15.-   Liu, G., et al., Evaluation of partially demineralized osteoporotic    cancellous bone matrix combined with human bone marrow stromal cells    for tissue engineering: an in vitro and in vivo study. Calcif Tissue    Int, 2008. 83(3): p. 176-85.-   Liu, G., et al., Evaluation of the viability and osteogenic    differentiation of cryopreserved human adipose-derived stem cells.    Cryobiology, 2008. 57(1): p. 18-24.

As discussed herein, human ASCs seeded bone substrates may becharacterized in terms of microstructure, cell number and cell identityusing histology, biochemical assay and flow cytometry. In an embodiment,these substrates may include bone material which has been previouslysubjected to a demineralization process.

FIG. 1 is a flow chart of a process for making an allograft with stemcells product. In an embodiment, a stromal vascular fraction may be usedto seed the allograft. It should be apparent from the present disclosurethat the term “seed” relates to addition and placement of the stem cellswithin, or at least in attachment to, the allograft, but is not limitedto a specific process. FIG. 2 illustrates a pellet of the stromalvascular fraction containing the desired stem cells.

In an exemplary embodiment, a method of combining mesenchymal stem cellswith a bone substrate is provided. The method may include obtainingadipose tissue having the mesenchymal stem cells together with unwantedcells. Unwanted cells may include hematopoietic stem sells and otherstromal cells. The method may further include digesting the adiposetissue to form a cell suspension having the mesenchymal stem cells andat least some or all of the unwanted cells. In another embodiment, thismay be followed by negatively depleting some of the unwanted cells andother constituents to concentrate mesenchymal stem cells.

Next, the method includes adding the cell suspension with themesenchymal stem cells to the bone substrate. This may be followed byculturing the mesenchymal stem cells and the bone substrate for a periodof time to allow the mesenchymal stem cells to adhere to the bonesubstrate. In order to provide a desired product, the method includesrinsing the bone substrate to remove the unwanted cells from the bonesubstrate.

In one embodiment, an allograft product may include a combination ofmesenchymal stem cells with a bone substrate such that the combinationis manufactured by the above exemplary embodiment.

In an embodiment, the adipose tissue may be obtained from a cadavericdonor. A typical donor yields 2 liters of adipose containing 18 millionMSCs. In one embodiment, a bone substrate may be from the same cadavericdonor as the adipose tissue. In another embodiment, the adipose tissuemay be obtained from a patient. In addition, both the bone substrate andthe adipose tissue may be obtained from the same patient. This mayinclude, but is not limited to, removal of a portion of the ilium (e.g.,the iliac crest) may be removed from the patient by a surgical procedureand adipose cells may be removed using liposuction. Other sources, andcombination of sources, of adipose tissue, other tissues, and bonesubstrates may be utilized.

Optionally, the adipose tissue may be washed prior to or duringdigestion. Washing may include using a thermal shaker at 75 RPM at 37°C. for at least 10 minutes. Washing the adipose tissue may includewashing with a volume of PBS substantially equal to the adipose tissue.In an embodiment, washing the adipose tissue includes washing with thePBS with 1% penicillin and streptomycin at about 37° C.

For example, washing the adipose tissue may include agitating the tissueand allowing phase separation for about 3 to 5 minutes. This may befollowed by aspirating off a infranatant solution. The washing mayinclude repeating washing the adipose tissue multiple times until aclear infranatant solution is obtained. In one embodiment, washing theadipose tissue may include washing with a volume of growth mediasubstantially equal to the adipose tissue.

In another exemplary embodiment, a method of combining mesenchymal stemcells with a bone substrate is provided. The method may includeobtaining bone marrow tissue having the mesenchymal stem cells togetherwith unwanted cells. Unwanted cells may include hematopoietic stem sellsand other stromal cells. The method may further include digesting thebone marrow tissue to form a cell suspension having the mesenchymal stemcells and the unwanted cells. In another embodiment, this may befollowed by naturally selecting MSCs and depleting some of the unwantedcells and other constituents to concentrate mesenchymal stem cells.

Next, the method includes adding the cell suspension with themesenchymal stem cells to the bone substrate. This may be followed byculturing the mesenchymal stem cells and the bone substrate for a periodof time to allow the mesenchymal stem cells to adhere to the bonesubstrate. In order to provide a desired product, the method includesrinsing the bone substrate to remove the unwanted cells from the bonesubstrate.

In one embodiment, an allograft product may include a combination ofmesenchymal stem cells with a bone substrate such that the combinationis manufactured by the above exemplary embodiment.

In another exemplary embodiment, a method of combining mesenchymal stemcells with a bone substrate is provided. The method may includeobtaining muscle tissue having the mesenchymal stem cells together withunwanted cells. Unwanted cells may include hematopoietic stem sells andother stromal cells. The method may further include digesting the muscletissue to form a cell suspension having the mesenchymal stem cells andthe unwanted cells. In another embodiment, this may be followed bynaturally selecting MSCs to concentrate mesenchymal stem cells.

Next, the method includes adding the cell suspension with themesenchymal stem cells to the bone substrate. This may be followed byculturing the mesenchymal stem cells and the bone substrate for a periodof time to allow the mesenchymal stem cells to adhere to the bonesubstrate. In order to provide a desired product, the method includesrinsing the bone substrate to remove the unwanted cells from the bonesubstrate.

In one embodiment, an allograft product may include a combination ofmesenchymal stem cells with a bone substrate such that the combinationis manufactured by the above exemplary embodiment.

In another exemplary embodiment, a method of combining mesenchymal stemcells with a bone substrate is provided. The method may includeobtaining tissue having the mesenchymal stem cells together withunwanted cells. Unwanted cells may include hematopoietic stem sells andother stromal cells. The method may further include digesting the tissueto form a cell suspension having the mesenchymal stem cells and at leastsome of the unwanted cells. In another embodiment, this may be followedby negatively depleting some of the unwanted cells and otherconstituents to concentrate mesenchymal stem cells.

Next, the method includes adding the cell suspension with themesenchymal stem cells to the bone substrate. In an embodiment, thissubstrate may include a bone material which has been subjected to ademineralization process. In another embodiment, this substrate may be anon-bone material, which may include (but is not limited to) a collagenbased material. This may be followed by culturing the mesenchymal stemcells and the bone substrate for a period of time to allow themesenchymal stem cells to adhere to the bone substrate. In order toprovide a desired product, the method includes rinsing the bonesubstrate to remove the unwanted cells from the bone substrate.

In one embodiment, an allograft product may include a combination ofmesenchymal stem cells with a bone substrate such that the combinationis manufactured by the above exemplary embodiment.

Digesting the cell suspension may include making a collagenase Isolution, and filtering the solution through a 0.2 μm filter unit,mixing the adipose tissue with the collagenase I solution, and addingthe cell suspension mixed with the collagenase I solution to a shakerflask. Digesting the cell suspension may further include placing theshaker with continuous agitation at about 75 RPM for about 45 to 60minutes so as to provide the adipose tissue with a visually smoothappearance.

Digesting the cell suspension may further include aspirating supernatantcontaining mature adipocytes so as to provide a pellet, which may bereferred to as a stromal vascular fraction. (See, for example, FIG. 2.)Prior to seeding, a lab sponge or other mechanism may be used to pat drybone substrate.

In one embodiment, adding the cell suspension with the mesenchymal stemcells to the bone substrate may include using a cell pellet for seedingonto the bone substrate. In an embodiment, adding the cell suspensionwith the mesenchymal stem cells to the bone substrate may include usinga cell pellet for seeding onto the bone substrate. In anotherembodiment, adding the cell suspension with the mesenchymal stem cellsto the bone substrate may include using a cell pellet for seeding ontothe bone substrate of cortical bone. In another embodiment, adding thecell suspension with the mesenchymal stem cells to the bone substratemay include adding the cell pellet onto the bone substrate of cancellousbone. In another embodiment, adding the cell suspension with themesenchymal stem cells to the bone substrate may include adding the cellpellet onto the bone substrate of ground bone. In another embodiment,adding the cell suspension with the mesenchymal stem cells to the bonesubstrate may include adding the cell pellet onto the bone substrate ofcortical/cancellous bone. In another embodiment, adding the cellsuspension with the mesenchymal stem cells to the bone substrate mayinclude adding the cell pellet onto the bone substrate of demineralizedcancellous bone.

In an embodiment, the method may include placing the bone substrate intoa cryopreservation media after rinsing the bone substrate. Thiscryopreservation media may be provided to store the final products. Forexample, the method may include maintaining the bone substrate into afrozen state after rinsing the bone substrate to store the finalproducts. The frozen state may be at about negative 80° C.

In another embodiment, Ficoll density solution may be utilized. Forexample, negatively depleting the concentration of the mesenchymal stemcells may include adding a volume of PBS and a volume of Ficoll densitysolution to the adipose solution. The volume of PBS may be 5 ml and thevolume of Ficoll density solution may be 25 ml with a density of 1.073g/ml. Negatively depleting the concentration of the mesenchymal stemcells may also include centrifuging the adipose solution at about 1160 gfor about 30 minutes at about room temperature. In one embodiment, themethod may include stopping the centrifuging the adipose solutionwithout using a brake.

Negatively depleting the concentration of the mesenchymal stem cells isoptional and may next include collecting an upper layer and an interfacecontaining nucleated cells, and discarding a lower layer of red cellsand cell debris. Negatively depleting the concentration of themesenchymal stem cells may also include adding a volume of D-PBS ofabout twice an amount of the upper layer of nucleated cells, andinverting a container containing the cells to wash the collected cells.Negatively depleting the concentration of the mesenchymal stem cells mayinclude centrifuging the collected cells to pellet the collected cellsusing the break during deceleration.

In an embodiment, negatively depleting the concentration of themesenchymal stem cells may further include centrifuging the collectedcells at about 900 g for about 5 minutes at about room temperature.Negatively depleting some of the unwanted cells may include discarding asupernatant after centrifuging the collected cells, and resuspending thecollected cells in a growth medium.

In one embodiment, adding the cell suspension with the mesenchymal stemcells to the bone substrate may include adding the cell pellet onto thebone substrate. Adding the solution with the mesenchymal stem cells tothe bone substrate may include adding cell pellet onto the bonesubstrate which was subjected to a demineralization process. In anotherembodiment, adding the cell suspension with the mesenchymal stem cellsto the bone substrate may include adding the cell pellet onto the bonesubstrate of cortical bone. In an embodiment, adding the cell suspensionwith the mesenchymal stem cells to the bone substrate includes addingthe cell pellet onto the bone substrate of cancellous bone. In anotherembodiment, adding the cell suspension with the mesenchymal stem cellsto the bone substrate may include adding the cell pellet onto the bonesubstrate of ground bone. In another embodiment, adding the cellsuspension with the mesenchymal stem cells to the bone substrate mayinclude adding the cell pellet onto the bone substrate ofcortical/cancellous bone. In another embodiment, adding the cellsuspension with the mesenchymal stem cells to the bone substrate mayinclude adding the cell pellet onto the bone substrate of demineralizedcancellous bone.

In an embodiment, the method may further include placing the bonesubstrate into a cryopreservation media after rinsing the bonesubstrate. This cryopreservation media may be provided to store thefinal products. The method may include maintaining the bone substrateinto a frozen state after rinsing the bone substrate to store the finalproducts. The frozen state may be at about negative 80° C.

The seeded allografts are cultured for a period of time to allow themesenchymal stem cells to adhere to the bone substrate. The unwantedcells were rinsed and removed from the bone substrate. After culturing,a lab sponge or other mechanism may be used to pat dry the bonesubstrate.

The mesenchymal stem cells are anchorage dependent. The mesenchymal stemcells naturally adhere to the bone substrate. The mesenchymal stem cellsare non-immunogenic and regenerate bone. The unwanted cells aregenerally anchorage independent. This means that the unwanted cellsgenerally do not adhere to the bone substrate. The unwanted cells may beimmunogenic and may create blood and immune system cells. For cellpurification during a rinse, mesenchymal stem cells adhere to the bonewhile unwanted cells, such as hematopoietic stem sells, are rinsed awayleaving a substantially uniform population of mesenchymal stem cells onthe bone substrate.

The ability to mineralize the extracellular matrix and to generate boneis not unique to MSCs. In fact, ASCs possess a similar ability todifferentiate into osteoblasts under similar conditions. Human ASCsoffer a unique advantage in contrast to other cell sources. Themultipotent characteristics of ASCs, as wells as their abundance in thehuman body, make these cells a desirable source in tissue engineeringapplications.

In various embodiments, bone substrates (e.g., cortical cancellousdowels, strips, cubes, blocks, discs, and granules, as well as othersubstrates formed in dowels, strips, cubes, blocks, discs, and granules)may be subjected to a demineralization process to remove blood, lipidsand other cells so as to leave a matrix. FIG. 3 illustrates variousexamples of strips (FIGS. 3A and 3B) and dowels (FIGS. 3C and 3D).Generally, these substrates may have a 3-D cancellous matrix structure,which MSCs may adhere to.

In addition, this method and combination product involve processing thatdoes not alter the relevant biological characteristics of the tissue.Processing of the adipose/stem cells may involve the use of antibiotics,cell media, collagenase. None of these affects the relevant biologicalcharacteristics of the stem cells. The relevant biologicalcharacteristics of these mesenchymal stem cells are centered on renewaland repair. The processing of the stem cells does not alter the cell'sability to continue to differentiate and repair.

In the absence of stimulation or environmental cues, mesenchymal stemcells (MSCs) remain undifferentiated and maintain their potential toform tissue such as bone, cartilage, fat, and muscle. Upon attachment toan osteoconductive matrix, MSCs have been shown to differentiate alongthe osteoblastic lineage in vivo. See, for example, the following, whichare incorporated by reference:

-   Arinzeh T L, Peter S J, Archambault M P, van den Bos C, Gordon S,    Kraus K, Smith A, Kadiyala S. Allogeneic mesenchymal stem cells    regenerate bone in a critical sized canine segmental defect. J Bone    Joint Surg Am. 2003; 85-A:1927-35.-   Bruder S P, Kurth A A, Shea M, Hayes W C, Jaiswal N, Kadiyala S.    Bone regeneration by implantation of purified, culture-expanded    human mesenchymal stem cells, J Orthop Res. 1998; 16:155-62.

Example 1 Adipose Recovery

Adipose was recovered from cadaveric donors. Adipose aspirate may becollected using liposuction machine and shipped on wet ice.

Washing

Adipose tissue was warmed up in a thermal shaker at RPM=75, 37° C. for10 min. Adipose was washed with equal volume of pre-warmed phosphatebuffered saline (PBS) at 37° C., 1% penicillin/streptomycin. Next, theadipose was agitated to wash the tissue. Phase separation was allowedfor about 3 to 5 minutes. The infranatant solution was aspirated. Thewash was repeated 3 to 4 times until a clear infranatant solution wasobtained.

The solution was suspended in an equal volume of growth media (DMEM/F12,10% FBS, 1% penicillin/streptomycin) and stored in a refrigerator atabout 4° C.

Digestion and Combining of Cell Suspension with Allografts

Digestion of the adipose was undertaken to acquire a stromal vascularfraction (SVF) followed by combining the solution onto an allograft.

Digestion involved making collagenase I solution, including 1% fetalbovine serum (FBS) and 0.1% collagenase I. The solution was filteredthrough a 0.2 um filter unit. This solution should be used within 1 hourof preparation.

Next, take out the washed adipose and mix with collagenase I solution at1:1 ratio. Mixture was added to a shaker flask.

The flask was placed in an incubating shaker at 37° C. with continuousagitation (at about RPM=75) for about 45 to 60 minutes until the tissueappeared smooth on visual inspection.

The digestate was transferred to centrifuge tubes and centrifuged for 5minutes at about 300-500 g at room temperature. The supernatant,containing mature adipocytes, was then aspirated. The pellet wasidentified as the stromal vascular fraction (SVF).

Growth media was added into every tube (i.e., 40 ml total was added intothe 4 tubes) followed by gentle shaking.

All of the cell mixtures were transferred into a 50 ml centrifuge tube.A 200 μl sample was taken, 50 μl is for initial cell count, and theremainder of the 150 μl was used for flow cytometry.

Aliquot cell mixtures were measured into 2 centrifuge tubes (of 10 mleach) and centrifuged at about 300 g for 5 minutes. The supernatant wasaspirated.

A cell pellet obtained from one tube was used for seeding ontoallografts. The allografts may include cortical/cancellous or both whichwas subjected to a demineralization process.

Certain volume of growth medium was added into the cell pellets andshaken to break the pellets. A very small volume of cell suspension wasadded onto allografts. After culturing in CO₂ incubator at 37° C. for afew hours, more growth medium (DMEM/F12, 10% FBS with antibiotics) wasadded. This was a static “seeding” process. A dynamic “seeding” processcan be used for particular bone substrate. 10 ml of a cell suspensionand bone substrate were placed in a 50 ml centrifuge tube on an orbitalshaker and agitated at 100 to 300 rpm for 6 hours.

After a few days (about 1 to 3 days), the allograft was taken out andrinsed thoroughly in PBS and sonicated to remove unwanted cells. Theallograft was put into cryopreservation media (10% DMSO, 90% serum) andkept frozen at −80° C. The frozen allograft combined with themesenchymal stem cells is a final product.

Example 2 Adipose Recovery

Adipose was recovered from cadaveric donors. Adipose aspirate may becollected using liposuction machine and shipped on wet ice.

Washing

Adipose tissue was processed in a thermal shaker at RPM=75, 37° C. for10 min. Adipose was washed with equal volume of pre-warmed phosphatebuffered saline (PBS) at 37° C., 1% penicillin/streptomycin. Next, theadipose was agitated to wash the tissue. Phase separation was allowedfor about 3 to 5 minutes. The supernatant solution was sucked off. Thewash was repeated 3 to 4 times until a clear infranatant solution wasobtained.

Acquire Ficoll Concentrated Stem Cells and Combine onto Allograft

Ficoll concentrated stem cells were acquired and seeded onto anallograft. 5 ml PBS was placed into the 50 ml tube with cells and 25 mlof 1.073 g/ml Ficoll density solution was added to the bottom of thetube with a pipet.

The tubes were subjected to centrifugation at 1160 g for 30 min at roomtemperature and stopped with the brake off. The upper layer andinterface, approximately 15 to 17 ml containing the nucleated cells werecollected with a pipet and transferred to a new 50 ml disposablecentrifuge tube. The lower layer contained red cells and cell debris andwas discarded.

Next, 2 volumes of D-PBS were added. The tubes were capped and mixgently by inversion to wash the cells.

The tubes with the diluted cells were then subjected to centrifugationat 900 g for 5 minutes at room temperature to pellet the cells with thebrake on during deceleration.

The supernatant was discarded and the washed cells were resuspended in10 ml of growth medium. 10 ml of growth media was added into the tubeand it was shaken gently A 1 ml sample was taken with 100 μl is for cellcount, and the remainder of 900 μl was used for flow cytometry.

The remainder of the cell mixtures were centrifuged at about 300 g forabout 5 minutes. The supernatant was aspirated.

A cell pellet was used for “seeding” onto allografts. Allografts mayinclude deminarealized bone, cortical/cancellous bone, or both. A verysmall volume of medium was added into the cell pellet and shaken. 100 μlof cell mixtures were added onto a 15 mm disc within a 24-well cultureplate.

After culturing the allograft in a CO2 incubator at about 37° C., 1 mlgrowth medium (DMEM/F12, 10% FBS with antibiotics) was added. This was astatic “seeding” process. A dynamic “seeding” process can be used for aparticular bone substrate.

After a few days (about 1 to 3 days), the allograft was taken out andrinsed thoroughly in PBS to remove unwanted cells. The allograft was putinto cryopreservation media (10% DMSO, 90% serum) and kept frozen at−80° C. The frozen allograft combined with the stem cells is a finalproduct.

Example 3 Bone Marrow Recovery

Adipose was recovered from cadaveric donors. Adipose aspirate may becollected using liposuction machine and shipped on wet ice.

Washing

The bone marrow sample is washed by adding 6 to 8 volumes of Dulbecco'sphosphate buffered saline (D-PBS) in a 50 ml disposable centrifuge,inverting gently and subjecting to centrifugation (800 g for 10 min) topellet cells to the bottom of the tube.

Acquire Stem Cells and Combine onto Allograft

The supernatant is discarded and the cell pellets from all tubes areresuspended in 1-2 ml of growth medium (DMEM, low glucose, with 10% FBSand 1% pen/strap). The cell mixtures are seeded onto allografts. With afew hours of culture in CO2 incubator at 37° C., more growth medium isadded. A few days later, the allograft is taken out and rinsedthoroughly in PBS and put into cryopreservation media (10% DMSO, 90%serum) and kept frozen.

Example 4 Skeletal Muscle Recovery

Skeletal muscle may be recovered from cadaveric donors.

Washing

Minced skeletal muscle (1-3 mm cube) is digested in a 3 mg/mlcollagenase D solution in α-MEM at 37° C. for 3 hours. The solution isfiltered with 100 um nylon mesh. The solution is centrifuged at 500 gfor 5 min.

Acquire Stem Cells and Combine onto Allograft

The supernatant is discarded and the cell pellets from all tubes areresuspended in 1-2 ml of growth medium (DMEM, low glucose, with 10% FBSand 1% pen/strap). The cell mixtures are seeded onto allografts. With afew hours of culture in CO2 incubator at 37° C., more growth medium willbe added. A few days later, the allograft is taken out and rinsedthoroughly in PBS and put into cryopreservation media (10% DMSO, 90%serum) and kept frozen.

Example 5 Adipose Recovery

Adipose was recovered from a cadaveric donor within 24 hours of deathand shipped in equal volume of DMEM in wet ice.

Washing

Adipose were washed 3 times with PBS and suspended in an equal volume ofPBS supplemented with Collagenase Type I prewarmed to 37° C. The tissuewas placed in a shaking water bath at 37° C. with continuous agitationfor 45 to 60 minutes and centrifuged for 5 minutes at room temperature.The supernatant, containing mature adipocytes, was aspirated. The pelletwas identified as the SVF (stromal vascular fraction).

Cortical Cancellous Bone Recovery

Human cortical cancellous bone was recovered from ilium crest from thesame donor. The samples were sectioned into strips (20×50×5 mm), andthen they were subjected to a demineralization process with HCl for 3hours, rinsed with PBS until the pH is neutral.

Digestion and Combining of Cell Suspension with Allograft

The adipose-derived stem cells (ASCs) were added onto the grafts andcultured in CO2 incubator at 37° C. Then the allografts were rinsedthoroughly in PBS to remove antibiotics and other debris. At the end,the allografts were put into cryopreservation media and kept frozen at−80° C.

Example 6 Adipose-Derived Stem Cell Characterization

Flow Cytometry Analysis

The following antibodies were used for flow cytometry. PE anti-CD73(clone AD2) Becton Dickinson, PE anti-CD90 (clone F15-42-1) AbD SeroTec,PE anti-CD105 (clone SN6) AbD SeroTec, PE anti-Fibroblasts/EpithelialCells (clone D7-FIB) AbD SeroTec, FITC anti-CD34 (clone 8G12) BectonDickinson, FITC anti-CD45 (clone 2D1) Becton Dickinson, and PEanti-CD271 (clone ME20.4-1.H4) Miltenyi BioTec. The Isotype controlswere FITC Mouse IgG1 Kappa (clone MOPC-21) Becton Dickinson, PE MouseIgG1 Kappa (clone MOPC-21) Becton Dickinson, and PE Mouse IgG2a Kappa(clone G155-178) Becton Dickinson.

A small aliquot of the cells were stained with a propidiumiodide/detergent solution and fluorescent nuclei were counted using ahemocytometer on a fluorescent microscope. This total cell count wasused to adjust the number of cells per staining tube to no more than5.0×105 cells. The cells were washed with flow cytometric wash buffer(PBS supplemented with 2% FBS and 0.1% NaN3), stained with the indicatedantibodies and washed again before acquisition. Staining was for 15minutes at room temperature (15-30C). At least 20,000 cells wereacquired for each sample on a FACScan flow cytometer equipped with a15-mW, 488-nm, argon-ion laser (BD Immunocytometry Systems, San Jose,Calif.). The cytometer QC and setup included running SpheroTech rainbow(3 μm, 6 peaks) calibration beads (SpheroTech Inc.) to confirminstrument functionality and linearity. Flow cytometric data werecollected and analyzed using CellQuest software (BD ImmunocytometrySystems). The small and large cells were identified by forward (FSC) andside-angle light scatter (SSC) characteristics. Autofluorescence wasassessed by acquiring cells on the flow cytometer without incubatingwith fluorochrome labeled antibodies. Surface antigen expression wasdetermined with a variety of directly labeled antibodies according tothe supplier's recommendations. Antibodies staining fewer than 20% ofthe cells relative to the Isotype-matched negative control wereconsidered negative (this is standard-of-practice for immunophenotypingleukocytes for leukemia lymphoma testing). The viability of the smalland large cells was determined using the Becton Dickinson Via-Probe(7-AAD).

In-Vitro Tri-Lineage Differentiation

Osteogenesis—Confluent cultures of primary ASCs were induced to undergoosteogenesis by replacing the stromal medium with osteogenic inductionmedium (Stempro® osteogenesis differentiation kit, Invitrogen). Cultureswere fed with fresh osteogenic induction medium every 3 to 4 days for aperiod of up to 3 weeks. Cells were then fixed in 10% neutral bufferedformalin and rinsed with DI water. Osteogenic differentiation wasdetermined by staining for calcium phosphate with Alizarin red (Sigma).

Adipogenesis—Confluent cultures of primary ASCs were induced to undergoadipogenesis by replacing the stromal medium with adipogenic inductionmedium (Stempro® adipogenesis differentiation kit, Invitrogen). Cultureswere fed with fresh adipogenic induction medium every 3 to 4 days for aperiod of up to 2 weeks. Cells were then fixed in 10% neutral bufferedformalin and rinsed with PBS. Adipogenic differentiation was determinedby staining for fat globules with oil red O (Sigma).

Chondrogenesis—Confluent cultures of primary ASCs were induced toundergo chondrogenesis by replacing the stromal medium with chondrogenicinduction medium (Stempro® chondrogenesis differentiation kit,Invitrogen). Cultures were fed with fresh chondrogenic induction mediumevery 3 to 4 days for a period of up to 3 weeks. Cells were then fixedin 10% neutral buffered formalin and rinsed with PBS. Chondrogenicdifferentiation was determined by staining for proteoglycans with Alcianblue (Sigma).

Final Product Characterization

Cell count may be preformed with a CCK-8 Assay. Cell Counting Kit 8(CCK-8, Dojindo Molecular Technologies, Maryland) allows sensitivecolorimetric assays for the determination of the number of viable cellsin cell proliferation assays. With reference to FIG. 4, there isillustrated a standard curve of total live ASCs using the CCK-8 assay.WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] is reduced bydehydrogenases in cells to give a yellow colored product (formazan),which is soluble in the tissue culture medium. The amount of theformazan dye generated by the activity of dehydrogenases in cells isdirectly proportional to the number of living cells. The allografts werethawed and rinsed with PBS and then patted dry. Growth medium and CCK-8solution were added into the allografts at a ratio of 10:1 cultured at37° C. for 2 hours and evaluated in a plate reader with excitation setto 460 nm and emission set to 650 nm. The results were interpolated froma standard curve (FIG. 1) based on ASCs only (passage=3).

Histology

When the cultures were terminated, the constructs were fixed in 10%neutral buffered formalin (Sigma, St. Louis, Mo.) for 48 h, put in aprocessor (Citadel 2000; Thermo Shandon, Pittsburgh, Pa.) overnight, andembedded in paraffin. Sections were cut to 8 μm and mounted onto glassslides and stained with hematoxylin and eosin (H&E). Conventional lightmicroscopy was used to analyze sections for matrix and cell morphology.

Statistical Analysis

All quantitative data were expressed as the mean±standard deviation.Statistical analysis was performed with one-way analysis of variance. Avalue of p≦0.05 was considered statistically significant.

Results

Final Product Appearance

FIG. 3 illustrates an appearance of strips, dowels and disks. In theseembodiments, all have a cortical bottom and cancellous top. Otherembodiments may be used.

ASC Characterization

Flow Cytometry—Immunophenotype of SVF

The SVF were stained with CD105, CD90 and CD73 to determine if therewere significant numbers of MSC present. The immunophenotype of thestromal vascular fraction was consistent from donor to donor. The largecells (mean 3%) have the following immunophenotype and mean percentage:D7-FIB+ (36%), CD105+ (43%), CD90+ (63%), CD73+ (28%) and CD34+ (62%).The small cells (mean 97%) contain only a small percentage of themarkers tested and therefore could not be immunophenotyped with thismethod: D7-FIB (5%), CD105 (6%), CD90 (15%), CD73 (6%) and CD34 (10%).The SVF contained a significant population of CD34+ cells (Large CD34+62% and small CD34+ 10%). The paucity of CD45+ cells (Large 15% andsmall 3%) would suggest that the SVF does not contain significantnumbers of WBC (CD45+, low FSC, low SSC) or hematopoietic stem cells(CD34+, low CD45+, medium FSC, low SSC). The anti-Fibroblasts/EpithelialCells (clone D7-FIB) antibody has been reported to be a good marker forMSC. The large cells were D7-FIB+36% and the small cells were D7-FIB+5%. CD271 should be negative on SVF cells and the large cells wereCD271+ 10% and the small cells were CD271+ 0%. Following adherence ofthe SVF (ASCs, P1), the immunophenotype became more homogenous for boththe large and small cells. The large cells (53%) have the followingimmunophenotype and percentage: D7-FIB+ (93%), CD105+ (98%), CD90+ (96%)and CD73+ (99%). The small cells (47%) have the followingimmunophenotype and percentage: D7-FIB+ (77%), CD105+ (75%), CD90+ (58%)and CD73+ (83%). The ASCs has lost CD34 marker expression (P3: large 4%and small 1%) (P1: large 8% and small 6%) and the CD45+ cells remainedlow (P3: large 2% and small 2%) (P1: large 3% and small 1%). This wouldsuggest that there are few WBC (CD45+, low FSC, low SSC) orhematopoietic stem cells (CD34+, low CD45+, medium FSC, low SSC)present. The anti-Fibroblasts/Epithelial Cell (clone D7-FIB) antibodyfor the adherent and cultured cells showed an increased expression. Thelarge cells were D7-FIB+ 93% and the small cells were D7-FIB+ 77%. CD271should become positive following adherence and culture of the SVF. ForP3 the large cells were CD271+ 4% and the small cells were CD271+ 1%.For P1 the large cells were CD271+ 27% and the small cells were CD271+3%. CD271 does not seem to be a useful marker for cultured MSC but moredata is required.

Estimated Mean Total Percentage of MSC

CD105 was chosen to estimate the mean total percentage of MSC; althoughthere is no single surface marker that can discern MSC in a mixedpopulation. For the SVF with a mean of 3% large cells, a mean of 43%CD105+ cells, the mean total percentage would be 1.3%. For the SVF witha mean of 98% small cells, a mean of 6% CD105+ cells, the mean totalpercentage would be 5.9%. Combining the large and small totals gives amean total of 7.2% MSC for the SVF.

In-vitro Tri-lineage Differentiation

FIG. 5 illustrates mineral deposition by ASCs cultured in osteogenicmedium (A) indicating early stages of bone formation. The samples werestained with alizarin red S. Negative controls (D) showed no sign ofbone formation. Fat globules seen in ASCs cultured in adipogenic medium(B) indicating differentiation into adipocytes. The samples were stainedwith Oil red O. The picture E is negative control. Proteoglycansproduced by ASCs cultured in chondrogenic medium (C) indicating earlystages of chondrogenesis. The samples were stained with alcian blue. Thenegative control (F) showed no sign of chondrogenesis.

For the osteogenic differentiation, morphological changes appearedduring the second week of the culture. At the end of the 21-dayinduction period, some calcium crystals were clearly visible. Celldifferentiation was confirmed by alizarin red staining (FIG. 3A).

The adipogenic potential was assessed by induction of confluent ASCs. Atthe end of the induction cycles (7 to 14 days), a consistent cellvacuolation was evident in the induced cells. Vacuoles brightly stainedfor fatty acid with oil red O staining (FIG. 3B). Chondrogenic potentialwas assessed by induction of confluent ASCs. At the end of the inductioncycles (14 to 21 days), the induced cells were clearly different fromnon-induced control cells. Cell differentiation was confirmed withAlcian blue staining (FIG. 3C).

Final Product Characterization

Cell count: CCK-8 Assay

28 grafts were tested from 8 donors and had an average of 50,000 livecells/graft.

Histology

H&E was performed to demonstrate cell morphology in relation to theunderlying substrate (cancellous bone matrix). The stem cells areelongated and adhere to the surface of cancellous bone. FIG. 6 is anillustration of H&E staining that showed that stem cells adhered to thebone surface.

CONCLUSIONS

The ability of DBM to enhance osteogenesis of ASCs in vitro and in vivois believed to be due to the interaction of osteoprogenitors with thesematrix incorporated osteoinductive factors, which can induce MSCs intoosteoblasts. In turn, the incorporation of an osteogenic cell sourceinto DBM can potentially limit the need for the migration and expansionof indigenous osteoprogenitors within defect sites, allowing for anincreased rate of bone formation and osseointegration. See, for example,the following, which are incorporated by reference:

-   Liu, G., et al., Evaluation of partially demineralized osteoporotic    cancellous bone matrix combined with human bone marrow stromal cells    for tissue engineering: an in vitro and in vivo study. Calcif Tissue    Int, 2008. 83(3): p. 176-85.-   Wang, J. and M. J. Glimcher, Characterization of matrix-induced    osteogenesis in rat calvarial bone defects: II. Origins of    bone-forming cells. Calcif Tissue Int, 1999. 65(6): p. 486-93.-   Wang, J. and M. J. Glimcher, Characterization of matrix-induced    osteogenesis in rat calvarial bone defects: I. Differences in the    cellular response to demineralized bone matrix implanted in    calvarial defects and in subcutaneous sites. Calcif Tissue    Int, 1999. 65(2): p. 156-65.-   Wang, J., et al., Characterization of demineralized bone    matrix-induced osteogenesis in rat calvarial bone defects: III. Gene    and protein expression. Calcif Tissue Int, 2000. 67(4): p. 314-20.-   Bruder, S. P. and B. S. Fox, Tissue engineering of bone. Cell based    strategies. Clin Orthop Relat Res, 1999(367 Suppl): p. S68-83.

Many studies have demonstrated that purified, culture-expanded humanMSCs can be directed into the osteogenic lineage in vitro, culminatingin a mineralized matrix production. See, for example, the following,which are incorporated by reference:

-   Chen, L. Q., et al., [Study of MSCs in vitro cultured on    demineralized bone matrix of mongrel]. Shanghai Kou Qiang Yi    Xue, 2007. 16(3): p. 255-8.-   Honsawek, S., D. Dhitiseith, and V. Phupong, Effects of    demineralized bone matrix on proliferation and osteogenic    differentiation of mesenchymal stem cells from human umbilical cord.    J Med Assoc That, 2006. 89 Suppl 3: p. S189-95.-   Kasten, P., et al., Ectopic bone formation associated with    mesenchymal stem cells in a resorbable calcium deficient    hydroxyapatite carrier. Biomaterials, 2005. 26(29): p. 5879-89.-   Qian, Y., Z. Shen, and Z. Zhang, [Reconstruction of bone using    tissue engineering and nanoscale technology]. Zhongguo Xiu Fu Chong    Jian Wai Ke Za Zhi, 2006. 20(5): p. 560-4.-   Liu, G., et al., Tissue-engineered bone formation with cryopreserved    human bone marrow mesenchymal stem cells. Cryobiology, 2008.    56(3): p. 209-15.-   Ko, E. K., et al., In vitro osteogenic differentiation of human    mesenchymal stem cells and in vivo bone formation in composite    nanofiber meshes. Tissue Eng Part A, 2008. 14(12): p. 2105-19.

The ability to mineralize the extracellular matrix and to generate boneis not unique to MSCs. In fact, ASCs possess a similar ability todifferentiate into osteoblasts under similar conditions. Human ASCsoffer a unique advantage in contrast to other cell sources. Themultipotent characteristics of ASCs, as wells as their abundance in thehuman body, make these cells a popular source in tissue engineeringapplications. This consistent cell-based new product has the potentialto be effective for bone regeneration.

1. A method of combining mesenchymal stem cells with a bone substrate,the method comprising: obtaining adipose tissue having the mesenchymalstem cells together with unwanted cells; digesting the adipose tissue toform a cell suspension having the mesenchymal stem cells and theunwanted cells; adding the cell suspension with the mesenchymal stemcells to seed the bone substrate so as to form a seeded bone substrate;culturing the mesenchymal stem cells on the seeded bone substrate for aperiod of time to allow the mesenchymal stem cells to adhere to the bonesubstrate; and rinsing the bone substrate to remove the unwanted cellsfrom the bone substrate.
 2. A method in accordance with claim 1, whereinthe obtaining the adipose tissue includes recovery from a cadavericdonor.
 3. A method in accordance with claim 1, wherein the bonesubstrate is from a cadaveric donor, and the obtaining the adiposetissue includes recovery from the same cadaveric donor as the bonesubstrate.
 4. A method in accordance with claim 1, wherein the obtainingthe adipose tissue includes recovery from a patient.
 5. A method inaccordance with claim 1, wherein the bone substrate is from a cadavericdonor, and the obtaining the adipose tissue includes recovery from thesame patient as the bone substrate.
 6. A method in accordance with claim1, wherein the digesting the adipose tissue includes making acollagenase I solution, and filtering the solution through a 0.2 μmfilter unit, mixing the adipose with the collagenase I solution, andadding the adipose with the collagenase I solution to a shaker flask. 7.A method in accordance with claim 6, wherein the digesting the adiposefurther includes placing the shaker with continuous agitation at about75 RPM for about 45 to 60 minutes so as to provide the adipose tissuewith a visually smooth appearance.
 8. A method in accordance with claim1, wherein the digesting the adipose further includes aspirating asupernatant containing mature adipocytes so as to provide a pellet.
 9. Amethod in accordance with claim 8, wherein the adding the suspensionwith the mesenchymal stem cells to the bone substrate includes addingthe cell suspension onto the bone substrate.
 10. A method in accordancewith claim 9, wherein the adding the suspension with the mesenchymalstem cells to the bone substrate includes adding the cell suspensiononto a bone substrate previously subjected to a demineralizationprocess.
 11. A method in accordance with claim 9, wherein the adding thesuspension with the mesenchymal stem cells to the bone substrateincludes adding the cell suspension onto cortical bone.
 12. A method inaccordance with claim 9, wherein the adding the suspension with themesenchymal stem cells to the bone substrate includes adding the cellsuspension onto cancellous bone.
 13. A method in accordance with claim9, wherein the adding the suspension with the mesenchymal stem cells tothe bone substrate includes adding the cell suspension onto ground bone.14. A method in accordance with claim 9, wherein the adding thesuspension with the mesenchymal stem cells to the bone substrateincludes adding the cell suspension onto cortical/cancellous bone.
 15. Amethod in accordance with claim 9, wherein the adding the suspensionwith the mesenchymal stem cells to the bone substrate includes addingthe cell suspension onto demineralized cancellous bone.
 16. A method inaccordance with claim 1, further including placing the bone substrateinto a cryopreservation media after rinsing the bone substrate to storethe final products.
 17. A method in accordance with claim 1, furtherincluding maintaining the bone substrate into a frozen state afterrinsing the bone substrate.
 18. A method in accordance with claim 17,wherein the frozen state is at about negative 80° C.
 19. A method inaccordance with claim 1, wherein the bone substrate includes a bonesubstrate previously subjected to a demineralization process.
 20. Amethod in accordance with claim 1, wherein the bone substrate includescortical bone.
 21. A method in accordance with claim 1, wherein the bonesubstrate includes cancellous bone.
 22. A method in accordance withclaim 1, wherein the bone substrate includes ground bone.
 23. A methodin accordance with claim 1, wherein the bone substrate includescortical/cancellous bone.
 24. A method in accordance with claim 1,wherein the bone substrate includes demineralized cancellous bone. 25.An allograft product including a combination of mesenchymal stem cellswith a bone substrate, and the combination manufactured by the method ofclaim
 1. 26. A method of combining mesenchymal stem cells with a bonesubstrate, the method comprising: obtaining adipose tissue having themesenchymal stem cells together with unwanted cells; digesting theadipose tissue to form a cell suspension having the mesenchymal stemcells and the unwanted cells to acquire a stromal vascular fraction, andthe digesting includes: making a collagenase I solution, and filteringthe solution through a 0.2 μm filter unit, mixing the adipose solutionwith the collagenase I solution, and adding the adipose solution mixedwith the collagenase I solution to a shaker flask; placing the shakerwith continuous agitation at about 75 RPM for about 45 to 60 minutes soas to provide the adipose tissue with a visually smooth appearance;aspirating a supernatant containing mature adipocytes so as to provide apellet; adding the cell suspension with the mesenchymal stem cells toseed the bone substrate by adding the cell suspension to seed the bonesubstrate so as to form a seeded bone substrate; culturing themesenchymal stem cells on the seeded bone substrate for a period of timeto allow the mesenchymal stem cells to adhere to the bone substrate; andrinsing the bone substrate to remove the unwanted cells from the bonesubstrate.
 27. An allograft product including a combination ofmesenchymal stem cells with a bone substrate, and the combinationmanufactured by the method of claim
 26. 28. A method of combiningmesenchymal stem cells with a bone substrate, the method comprising:obtaining tissue having the mesenchymal stem cells together withunwanted cells; digesting the tissue to form a cell suspension havingthe mesenchymal stem cells and the unwanted cells; adding the cellsuspension with the mesenchymal stem cells to seed the bone substrate soas to form a seeded bone substrate; culturing the mesenchymal stem cellson the seeded bone substrate for a period of time to allow themesenchymal stem cells to adhere to the bone substrate; and rinsing thebone substrate to remove the unwanted cells from the bone substrate. 29.A method in accordance with claim 28, wherein the obtaining the tissueincludes recovery from a cadaveric donor.
 30. A method in accordancewith claim 28, wherein the bone substrate is from a cadaveric donor, andthe obtaining the tissue includes recovery from the same cadaveric donoras the bone substrate.
 31. A method in accordance with claim 28, whereinthe obtaining the tissue includes recovery from a patient.
 32. A methodin accordance with claim 28, wherein the bone substrate is from acadaveric donor, and the obtaining the tissue includes recovery from thesame patient as the bone substrate.
 33. A method in accordance withclaim 28, wherein the bone substrate includes a bone substratepreviously subjected to a demineralization process.
 34. A method inaccordance with claim 28, wherein the bone substrate includes corticalbone.
 35. A method in accordance with claim 28, wherein the bonesubstrate includes cancellous bone.
 36. A method in accordance withclaim 28, wherein the bone substrate includes ground bone.
 37. A methodin accordance with claim 28, wherein the bone substrate includescortical/cancellous bone.
 38. A method in accordance with claim 28,wherein the bone substrate includes demineralized cancellous bone. 39.An allograft product including a combination of mesenchymal stem cellswith a bone substrate, and the combination manufactured by the method ofclaim
 28. 40. A method of combining mesenchymal stem cells with a bonesubstrate, the method comprising: obtaining bone marrow tissue havingthe mesenchymal stem cells together with unwanted cells; digesting thebone marrow tissue to form a cell suspension having the mesenchymal stemcells and the unwanted cells; adding the cell suspension with themesenchymal stem cells to seed the bone substrate so as to form a seededbone substrate; culturing the mesenchymal stem cells on the seeded bonesubstrate for a period of time to allow the mesenchymal stem cells toadhere to the bone substrate; and rinsing the bone substrate to removethe unwanted cells from the bone substrate.
 41. A method in accordancewith claim 40, wherein the obtaining the bone marrow tissue includesrecovery from a cadaveric donor.
 42. A method in accordance with claim40, wherein the bone substrate is from a cadaveric donor, and theobtaining the bone marrow tissue includes recovery from the samecadaveric donor as the bone substrate.
 43. A method in accordance withclaim 40, wherein the obtaining the bone marrow tissue includes recoveryfrom a patient.
 44. A method in accordance with claim 40, wherein thebone substrate is from a cadaveric donor, and the obtaining the bonemarrow tissue includes recovery from the same patient as the bonesubstrate.
 45. A method in accordance with claim 40, wherein the bonesubstrate includes a bone substrate previously subjected to ademineralization process.
 46. A method in accordance with claim 40,wherein the bone substrate includes cortical bone.
 47. A method inaccordance with claim 40, wherein the bone substrate includes cancellousbone.
 48. A method in accordance with claim 40, wherein the bonesubstrate includes ground bone.
 49. A method in accordance with claim40, wherein the bone substrate includes cortical/cancellous bone.
 50. Amethod in accordance with claim 40, wherein the bone substrate includesdemineralized cancellous bone.
 51. An allograft product including acombination of mesenchymal stem cells with a bone substrate, and thecombination manufactured by the method of claim
 40. 52. A method ofcombining mesenchymal stem cells with a bone substrate, the methodcomprising: obtaining muscle tissue having the mesenchymal stem cellstogether with unwanted cells; digesting the muscle tissue to form a cellsuspension having the mesenchymal stem cells and the unwanted cells;adding the cell suspension with the mesenchymal stem cells to seed thebone substrate so as to form a seeded bone substrate; culturing themesenchymal stem cells on the seeded bone substrate for a period of timeto allow the mesenchymal stem cells to adhere to the bone substrate; andrinsing the bone substrate to remove the unwanted cells from the bonesubstrate.
 53. A method in accordance with claim 52, wherein theobtaining the muscle tissue includes recovery from a cadaveric donor.54. A method in accordance with claim 52, wherein the bone substrate isfrom a cadaveric donor, and the obtaining the muscle tissue includesrecovery from the same cadaveric donor as the bone substrate.
 55. Amethod in accordance with claim 52, wherein the obtaining the muscletissue includes recovery from a patient.
 56. A method in accordance withclaim 52, wherein the bone substrate is from a cadaveric donor, and theobtaining the muscle tissue includes recovery from the same patient asthe bone substrate.
 57. A method in accordance with claim 52, whereinthe bone substrate includes demineralized bone.
 58. A method inaccordance with claim 52, wherein the bone substrate includes corticalbone.
 59. A method in accordance with claim 52, wherein the bonesubstrate includes cancellous bone.
 60. A method in accordance withclaim 52, wherein the bone substrate includes ground bone.
 61. A methodin accordance with claim 52, wherein the bone substrate includescortical/cancellous bone.
 62. A method in accordance with claim 52,wherein the bone substrate includes demineralized cancellous bone. 63.An allograft product including a combination of mesenchymal stem cellswith a bone substrate, and the combination manufactured by the method ofclaim 52.